Lactosamine oligosaccharide and method for producing the same

ABSTRACT

A method for producing an N-acetyllactosamine oligosaccharide, comprising the steps of: 
     adjusting a sulfate group content of keratan sulfate; 
     allowing an enzyme having an ability to cleave a glycosidic linkage of keratan sulfate to act on the keratan sulfate with the adjusted sulfate group content to obtain a sulfated N-acetyllactosamine oligosaccharide; and 
     completely desulfating said sulfated N-acetyllactosamine oligosaccharide.

This application is a divisional of prior application Ser. No.09/230,128 filed Jan. 22, 1999, now U.S. Pat. No. 6,132,994, which is a371 of PCT/JP97/02551, filed Jul. 23, 1997. The complete disclosure ofthis previous application is hereby incorporated by this referencethereto.

TECHNICAL FIELD

The present invention relates to a novel method for producing anN-acetyllactosamine oligosaccharide and more particularly to a methodfor producing an N-acetyllactosamine oligosaccharide by enzymaticreaction and chemical reaction from keratan sulfate whose sulfate groupcontent has been adjusted. Also, the present invention relates to anovel method for producing a sulfated N-acetyllactosamineoligosaccharide. Further, the present invention relates to a novelN-acetyllactosamine oligosaccharide.

BACKGROUND ART

Oligosaccharides having an N-acetyllactosamine (LacNAc) structure astheir skeleton and derivatives thereof play an important physiologicalrole in vivo as a sugar chain having ability to bind to selectin familycompounds. It has been revealed that expression of these sugar chains oncell surfaces participates in specific adhesion between heterogeneouscells, and function activators or inhibitors utilizing these sugarchains are expected to provide useful drugs. That is, sialyl Lewis X(SLe^(x)), which is a representative compound, and analogues thereof areconsidered to be useful for alleviating various diseases accompaniedwith inflammation by blocking selectin molecules to inhibit the adhesionof lymphocytes to endothelial cells. Further, these sugar chains areconsidered to participate in adhesion of cancer cells to endothelialcells when the cancer cells undergo blood circulatory metastasis oradhesion of microorganisms to target cells when the microorganismsinfect to the target cells, and therefore attached importance as a rawmaterial for developing drugs for suppressing these diseases.

The oligosaccharides having an N-acetyllactosamine structure as theirskeleton and derivatives thereof are expected to play an important rolein the development of novel drugs and in the research of physiologicalactivity in vivo. If there is obtained an N-acetyllactosamineoligosaccharide, which is their basic skeleton, in particular a longchain N-acetyllactosamine oligosaccharide similar to natural-occurringligand sugar chains of selectin family, it is considered possible tosynthesize the ligand sugar chains of selectin family, which occurs in atrace amount in a natural state, by adding properly a sialic acidresidue, a fucosyl residue, a sulfate group, ceramide or the like to theN-acetyllactosamine oligosaccharide.

Further, stable supply of N-acetyllactosamine oligosaccharides, inparticular long chain N-acetyllactosamine oligosaccharides, is desired.However, oligosaccharides that have N-acetyllactosamine structure astheir skeleton exist only in a trace amount in a natural state and it isvery difficult to synthesize long chain N-acetyllactosamineoligosaccharides so that a method for producing N-acetyllactosamineoligosaccharides on an industrial scale and at low costs is desired.

Also, sulfated N-acetyllactosamine oligosaccharides, in particular longchain sulfated N-acetyllactosamine oligosaccharides, are useful asintermediates for the synthesis of N-acetyllactosamine oligosaccharides,in particular long chain N-acetyllactosamine oligosaccharides and,hence, it is expected that provision of a method for producing sucholigosaccharides will lead to stable supply of N-acetyllactosamineoligosaccharides.

DISCLOSURE OF THE INVENTION

The present invention has been made from the above-described viewpoints,and its object is to provide a novel N-acetyllactosamine oligosaccharidewhich serves as a raw material for the synthesis of the ligand sugarchains of selectin family, a novel method for producing anN-acetyllactosamine oligosaccharide, and a novel method for producing asulfated N-acetyllactosamine oligosaccharide.

As a result of intensive investigation by the present inventors withview to achieving the above-described object, it has now been found thatan N-acetyllactosamine oligosaccharide (in particular a long chainN-acetyllactosamine oligosaccharide) can be obtained by adjusting thesulfate group content of keratan sulfate having an N-acetyllactosaminestructure as a basic skeleton, enzymatically degrading the keratansulfate with the thus adjusted sulfate group content to sulfatedN-acetyllactosamine oligosaccharides, and then completely desulfatingthe sulfated N-acetyllactosamine oligosaccharides. The present inventionhas been completed based on this finding.

It has also been found that a sulfated N-acetyllactosamineoligosaccharide (in particular a long chain sulfated N-acetyllactosamineoligosaccharide) can be obtained by adjusting the sulfate group contentof keratan sulfate and enzymatically digesting the keratan sulfate withthe adjusted sulfate group content and the present invention has beencompleted based on this finding.

Further, it has been successful in obtaining a novel N-acetyllactosamineoligosaccharide, which has led to the present invention.

Thus, the present invention provides a method for producing anN-acetyllactosamine oligosaccharide, comprising the steps of: adjustinga sulfate group content of keratan sulfate, allowing an enzyme having anactivity to cleave a glycosidic linkage of keratan sulfate (hereinsometimes referred to simply as “keratan sulfate-degrading enzyme”) toact on the keratan sulfate with the adjusted sulfate group content toobtain a sulfated N-acetyllactosamine oligosaccharide, and thencompletely desulfating the sulfated N-acetyllactosamine oligosaccharide(herein sometimes referred to simply as “method 1 of the presentinvention”), and a novel N-acetyllactosamine oligosaccharide (hereinsometimes referred to simply as “oligosaccharide of the presentinvention”).

Also, the present invention provides a method for producing a sulfatedN-acetyllactosamine oligosaccharide, comprising at least the steps of:adjusting a sulfate group content of keratan sulfate, and allowing oneor more enzymes belonging to any one of the enzyme groups of three typesof keratan sulfate-degrading enzymes each having a different substratespecificity depending on presence or absence of a sulfate group to acton the keratan sulfate with the adjusted sulfate group content (hereinsometimes referred to simply as “method 2 of the present invention”).Note that sometimes the methods 1 and 2 of the present invention arecollectively referred to simply as “method of the present invention”.

The term “N-acetyllactosamine” as used herein refers to a structure(Gal-GlcNAc) containing a galactose residue (Gal) and anN-acetylglucosamine residue (GlcNAc) linked through a glycosidiclinkage. Also, the term “N-acetyllactosamine” as used herein includes astructure (GlcNAc-Gal) containing an N-acetylglucosamine residue(GlcNAc) and a galactose residue (Gal) are linked through a glycosidiclinkage.

The term “N-acetyllactosamine oligosaccharide” as used herein refers toan oligosaccharide having at least one N-acetyllactosamine structure,and includes, for example, N-acetyllactosamine itself andoligosaccharides containing N-acetyllactosamine units linked one afteranother repeatedly through a glycosidic linkage. Also, a sialic acidresidue may be added to its non-reducing ends. The non-reducing end andreducing end may be either a galactose residue or an N-acetylglucosamineresidue. These are both embraced by the term “N-acetyllactosamineoligosaccharide” used herein. That is, the oligosaccharides representedby the following general formulae (1) to (6) are encompassed by the“N-acetyllactosamine oligosaccharide” used herein:

(GlcNAc-Gal)_(n)  (1)

SA-Gal-(GlcNAc-Gal)_(m)  (2)

(Gal-GlcNAc)_(n)  (3)

SA-(Gal-GlcNAc)_(m)  (4)

Gal-(GlcNAc-Gal)_(n)  (5)

GlcNAc-(Gal-GlcNAc)_(n)  (6)

wherein, Gal represents a galactose residue, GlcNAc represents anN-acetylglucosamine residue, SA represents a sialic acid residue, —represents a glycosidic linkage, n is an integer of 1 to 6, and m is aninteger of 1 to 10.

The term “sulfated N-acetyllactosamine oligosaccharide” as used hereinrefers to an N-acetyllactosamine oligosaccharide having a sulfate group.

The term “sulfated oligosaccharide” as used herein refers to anoligosaccharide having a sulfate group.

The term “adjusting a sulfate group content” as used herein refers toincreasing a sulfate group content (i.e., sulfating), decreasing asulfate group content (i.e., partially desulfating), and unchanging asulfate group content if a desired sulfate group content has alreadybeen reached without further increasing or decreasing it.

The term “partially desulfating” as used herein refers to removingpartially sulfate groups and differs from “completely desulfating” inthat the former allows a part of sulfate groups to remain.

The term “completely desulfating” as used herein refers to removingsubstantially all the sulfate groups, thus differing from partiallyremoving sulfate groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the relationship between the time ofpartial desulfation of keratan sulfate by a methanol-hydrochloric acidmethod and the yield of sulfated N-acetyllactosamine oligosaccharides of6- to 12-saccharides.

FIG. 2 is a graph illustrating the relationship between the temperatureof partial desulfation of keratan sulfate by a DMSO method and the yieldof sulfated N-acetyllactosamine oligosaccharides of 6- to12-saccharides.

FIG. 3 is an illustration of elution curves of gel filtration ofproducts obtained by allowing endo-β-galactosidase from Escherichiafreundii or keratanase II to act on keratan sulfate partially desulfatedby a DMSO method at various temperatures.

BEST MODE FOR CARRYING OUT THE INVENTION 1. Method of the PresentInvention

The method 1 of the present invention is a method for producing anN-acetyllactosamine oligosaccharide, which comprises the steps of:adjusting a sulfate group content of keratan sulfate, allowing a keratansulfate-degrading enzyme to act on the keratan sulfate with the adjustedsulfate group content to obtain a sulfated N-acetyllactosamineoligosaccharide, and then completely desulfating the sulfatedN-acetyllactosamine oligosaccharide.

The method 2 of the present invention is a method for producing asulfated N-acetyllactosamine oligosaccharide, which comprises the stepsof: adjusting a sulfate group content of keratan sulfate, and allowingone or more enzymes belonging to any one of the enzyme groups of threetypes of keratan sulfate-degrading enzymes each having a differentsubstrate specificity depending on presence or absence of a sulfategroup to act on the keratan sulfate with the adjusted sulfate groupcontent.

Hereafter, the technology involved in each step of the method of thepresent invention will be described in detail. Items (1) to (5)described below are technologies common to both the methods 1 and 2 ofthe present invention and items (6) and (7) are related to the method 1of the present invention.

(1) Keratan Sulfate Used in the Method of the Present Invention

Keratan sulfate is a glycosaminoglycan having as a repeating unit adisaccharide (i.e., N-acetyllactosamine; LacNAc) consisting of agalactose residue (Gal) and an N-acetylglucosamine residue (GlcNAc)linked through a glycosidic linkage and having a sulfate group in aratio of 1 to 2 moles of sulfate group per mole of the disaccharide. Thesulfate group content of keratan sulfate differs depending on animalspecies and organ. Usually, keratan sulfate is produced from rawmaterials, for example, cartilage, bones, corneas, etc., ofcartilaginous fish such as shark, and mammals such as whales and oxen.The keratan sulfate that can be used in the method of the presentinvention may be any generally available one and is not limited to aparticular one. However, it is preferred to use a highly sulfatedkeratan sulfate in which galactose residues of constituent sugars aresulfated (highly sulfated keratan sulfate containing 1.5 to 2 moles ofsulfate group per mole of the constituent disaccharide is sometimesreferred to as keratan polysulfate). Sulfate groups are attached to thegalactose residue preferably at the 6-position. Such a highly sulfatedkeratan sulfate can be obtained, for example, from proteoglycan of thecartilage of cartilaginous fish such as shark. Alternatively,commercially available ones may also be used. The term “keratan sulfate”as used herein includes highly sulfated keratan sulfate and keratanpolysulfate unless otherwise indicated.

(2) Adjustment of the Sulfate Group Content of Keratan Sulfate

The method for adjusting the sulfate group content of keratan sulfateincludes sulfating and partially desulfating until a desired sulfategroup content is reached, and unchanging a sulfate group content if adesired sulfate group content has already been reached without furtherincreasing or decreasing it. One having ordinary skill in the art mayselect the method for adjusting the sulfate group content appropriatelydepending the sulfate group content of keratan sulfate to be used in themethod of the present invention, the type of keratan sulfate-degradingenzyme to be used in the method of the present invention, and the targetsize of an oligosaccharide.

The sulfate group content is adjusted to such an extent thatoligosaccharides having a target molecular size can be obtained by theaction of a keratan sulfate-degrading enzyme and more specifically, toan extent that sulfate groups are present in a ratio of 0.3 to 1.5 moleson average per mole of the constituent disaccharide.

When an enzyme selected from the group consisting of the enzyme groups(1) and (2) as explained in detail below is used as the keratansulfate-degrading enzyme, it is preferred to use keratan sulfate havinga sulfate group content which has been adjusted such that a sulfategroup is present in a ratio of 1.1 to 1.5 moles per mole of theconstituent disaccharide. Also, when an enzyme selected from the groupconsisting of the enzyme groups (2) and (3) as explained in detail belowis used as the keratan sulfate-degrading enzyme, it is preferred to usekeratan sulfate having a sulfate group content which has been adjustedsuch that a sulfate group is present in a ratio of 0.3 to 0.8 mole permole of the constituent disaccharide.

Hereafter, the method for adjusting a sulfate group content of keratansulfate will be described.

(2-1) Partial Desulfation

Partial desulfation may be performed by a known method for desulfationof glycosaminoglycan. However, it is necessary to set up the reactionconditions such as the temperature and time of desulfation reaction, andthe concentration of a desulfating agent and the like lest substantiallyall the sulfate groups should be removed. More particularly, thetemperature and time of desulfation reaction as well as theconcentration of a desulfating agent and the like are set up at lowervalues than the reaction conditions at which substantially all thesulfate groups are removed. One skilled in the art may appropriatelyselect specific reaction conditions depending on the keratan sulfate tobe used, type of the keratan sulfate-degrading enzyme to be used incleaving the glycosidic linkages of keratan sulfate with an adjustedsulfate group content, target molecular size (molecular weight, numberof sugar chains) of N-acetyllactosamine oligosaccharide and the like.

The known method for desulfation of glycosaminoglycan includes, forexample, acid hydrolysis, alkaline decomposition, and heating in anorganic solvent (Method in Carbohydrate Chemistry, vol. VIII, p281-289(1980)). That is, as the desulfating agent, there can be used an acid,an alkali, an organic solvent and the like.

In the case of acid hydrolysis, for example, there can be used as theacid, an inorganic acid, an organic acid, a highly acidic cationexchange resin, and a particularly sulfonated resin. As the reactionsolvent, there can be used water, methanol and the like. In the methodof the present invention, it is preferred to use anhydrous methanolsolution containing acetyl chloride.

In the case of using an organic solvent, for example, the organicsolvent may be an aprotic solvent such as dimethyl sulfoxide (DMSO),N,N-dimethylformamide (DMF), and pyridine. When DMSO is used, a methodin which keratan sulfate pyridinium salt is heated at 20 to 100° C. in aDMSO solution containing 5 to 10% of water or methanol is widely used(Shin Seikagaku Jikken Koza, vol. 3, Toshitsu II, Tokyo Kagaku Dojin,p325-326 (1989)) and it is preferred to use this method in the method ofthe present invention.

Also, a desulfation method in which a silylating agent is used may beemployed.

Japanese Patent Application Laid-open No. 5-230090 (1993) discloses amethod for producing a desulfated sugar in which an organic base salt ofa sulfated sugar is reacted with an N,O-bis(trimethylsilyl)acetamide inthe presence of the organic base to selectively desulfate sulfate groupsattached to primary hydroxyl groups of the sulfated sugar. This methodmay also be used in the method of the present invention.

First, keratan sulfate is dissolved in an organic base as a solvent toform an organic base salt of keratan sulfate. Examples of the organicbase salt of keratan sulfate include salts of aromatic amines such aspyridine, dimethylaniline, and diethylaniline; of tertiary amines suchas trimethylamine, triethylamine, tributylamine,N,N-diisopropylethylamine, trioctylamine, andN,N,N′,N′-tetramethyl-1,8-naphthalenediamine; and of N-alkylheterocyclic amines such as N-methylpyrimidine, N-ethylpyrimidine,N-methylmorpholine, and N-ethylmorpholine.

Subsequently, desulfation can be carried out at a reaction temperatureof room temperature to 100° C. in the presence of anhydrous organic basewith adding a trimethylsilylating agent such asN,O-bis(trimethylsilyl)acetamide (BTSA),N,O-bis(trimethylsilyl)trifluoroacetamide (BTSFA),N,O-bis(trimethylsilyl)difluoroacetamide,N,O-bis(trimethylsilyl)monofluoroacetamide or the like.

Japanese Patent Application Laid-open No. 7-62001 (1995) discloses amethod for producing a desulfated sugar characterized by subjecting asulfated sugar to desulfation reaction in the presence of a silylatingagent represented by the general formula (A):

CH₃—C[—OSi(R )₃]═CH—COCH₃  (A)

wherein, R, which may be the same or different, represent independentlyan alkyl group or an aryl group. This method may also be used in themethod of the present invention. Even in this method, it is preferred toallow the silylating agent represented by general formula (A) above toreact with the organic base salt of sulfated sugar. As the organic basesalt, there can be used the same organic base salts as described inJapanese Patent Application Laid-open No. 5-230090 (1993) supra.Examples of (R)₃SiO in general formula (A) above includetrimethylsilyloxy, triethylsilyloxy, dimethylisopropylsilyloxy,isopropyldimethylsilyloxy, methyldi-t-butylsilyloxy,t-butyldimethylsilyloxy, t-butyldiphenylsilyloxy, andtriisopropylsilyloxy. The most preferred silylating agent represented bythe general formula (A) above is 2-trimethylsiloxypent-2-en-4-on.Desulfation can be performed by conducting reaction at room temperatureto 100° C.

International Publication (WO96/01278) discloses a method for producinga desulfated polysaccharide characterized by selectively desulfatingsulfate groups attached to the primary hydroxyl groups of thepolysaccharide by allowing a silylating agent represented by the generalformula (B):

(R¹)₃C—C(═O)N(—R²)—Si(R³)₃  (B)

wherein, R¹, which may be the same or different, represent independentlya hydrogen atom or a halogen atom, R² represents a lower alkyl group,and R³, which may be the same or different, represent independently alower alkyl group, an aryl group, or a halogen atom, to react therewith.This method is also usable in the method of the present invention. Inthis method too, it is preferred to react the silylating agentrepresented by the above-described formula with the organic base salt ofsulfated sugar. As the organic base salt of sulfate sugar, there can beused the same organic base salts as described in Japanese PatentApplication Laid-open No. 5-230090 (1993) supra. As the silylatingagent, there can be used those represented by general formula (B) abovein which R¹, which may be the same or different, represent independentlya hydrogen atom or a halogen atom such as fluorine, R² represents alower alkyl group having 1 to 6 carbon atoms, such as a methyl group, anethyl group, a propyl group, an isopropyl group, a butyl group, anisobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group,an isopentyl group, and a hexyl group, and R³, which may be the same ordifferent, represent independently the same lower alkyl group asdescribe above, an aryl group such as a phenyl group, or a halogen atomsuch as chlorine or fluorine. Examples of (R³) ₃Si in general formula(B) above include trimethylsilyl, triethylsilyl, dimethylisopropylsilyl,isopropyldimethylsilyl, methyldi-t-butylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, and triisopropylsilyl. Most preferred silylatingagents represented by general formula (B) above includeN-methyl-N-trimethylsilylacetamide (MTMSA) andN-methyl-N-trimethylsilyltrifluoroacetamide (MTSTFA). Desulfation can beperformed by conducting reaction at room temperature to 100° C.

Furthermore, an enzyme having an activity to desulfate keratan sulfatemay be used.

Appropriately setting up reaction conditions in the known methods fordesulfating glycosaminoglycan can give rise to keratan sulfate with theamount of Sulfate Group Reduced to a Suitable Level.

(2-2) Sulfation

Sulfation can be carried out by a known method for sulfatingglycosaminoglycan and is not limited to a particular method. Forexample, there can be cited the method disclosed in Japanese PatentApplication Laid-open No. 61-47701 (1986). As stated above, keratansulfate generally has a high sulfate group content and therefore it ismostly the case that the sulfate group content of keratan sulfate isadjusted by partial desulfation. However, when keratan sulfate or thelike that has an extremely low sulfate group content is available andusable, the sulfate group content may be adjusted by sulfation. Whensulfation is used for adjusting the sulfate group content, it ispreferred to sulfate only the 6-position of a galactose residue and/orN-acetylglucosamine residue, so that it is preferred to select asulfation method that is specific to the 6-position.

Also, an enzyme which transfers a sulfate group specifically to the6-position of galactose residue and/or N-acetylglucosamine residue ofkeratan sulfate (sulfotransferase) may be used.

The sulfate group content of keratan sulfate or that with an adjustedsulfate group content can be determined by a known method and evaluated,for example, based on the results obtained by allowing a keratansulfate-degrading enzyme thereon. Alternatively, it can be evaluatedbased on the mole number of sulfate group per mole of the constituentdisaccharide. The mole number of sulfate group per mole of theconstituent disaccharide can be determined by determining galactose orN-acetylglucosamine by an anthrone method or aminosugar analysis toobtain number of sugar residues, determining a sulfate group content byion chromatography of hydrochloric acid decomposition product of asugar, and calculating from these values. If the keratan sulfate used asa raw material has a desired sulfate group content, there is no need forconducting adjustment of the sulfate group content (i.e., sulfation orpartial desulfation) positively but the keratan sulfate can be used asit is in the invention.

In the method of the present invention, since keratan sulfate generallyhas a high sulfate group content and it is preferred to use highlysulfated keratan sulfate or keratan polysulfate or for some otherreasons, the adjustment of a sulfate group content of keratan sulfate inthe method of the present invention is in many cases carried out bypartial desulfation, which is preferred. It is preferred to accordinglyselect a keratan sulfate-degrading enzyme (described later on).

(3) Keratan Sulfate-degrading Enzyme Used in the Method of the PresentInvention

The keratan sulfate-degrading enzyme that can be used in the method ofthe present invention is not limited to a particular one as far as it isan enzyme that has an activity to cleave the glycosidic linkage ofkeratan sulfate. However, it is preferred to use one or more enzymesbelonging to any one of enzyme groups (1) to (3) below.

Enzyme group (1): Enzymes having the following activity and substratespecificity:

(a) Activity:

The enzyme cleaves a β-galactosidic linkage of keratan sulfate.

(b) Substrate Specificity:

If a galactose residue participating in the β-galactosidic linkage has asulfate group at its 6-position, the enzyme does not act on theβ-galactosidic linkage.

Whether or not a sulfate group is present at 6-position of anN-acetylglucosamine residue adjacent to a non-reducing end of agalactose residue participating in the β-galactosidic linkage and havingno sulfate group at its 6-position, the enzyme acts on theβ-galactosidic linkage.

The enzyme group (1) preferably comprises endo-β-galactosidases.

The enzymes of the enzyme group (1) include, for example, anendo-β-galactosidase from Escherichia freundii (H. Nakagawa, T. Yamada,J-L. Chien, A. Gardas, M. Kitamikado, S-C. Li, Y-T. Li, J. Biol. Chem.,255, 5955 (1980); herein sometimes referred to simply as “E-Galase”),which is preferred.

Enzyme group (2): Enzymes having the following activity and substratespecificity:

(a) Activity:

The enzyme cleaves a β-galactosidic linkage of keratan sulfate.

(b) Substrate Specificity:

If a galactose residue participating in the β-galactosidic linkage has asulfate group at its 6-position, the enzyme does not act on theβ-galactosidic linkage.

If a sulfate group is present at 6-position of an N-acetylglucosamineresidue adjacent to a non-reducing end of a galactose residueparticipating in the β-galactosidic linkage and having no sulfate groupat its 6-position, the enzyme acts on the β-galactosidic linkage.

If a sulfate group is absent at the 6-position of theN-acetylglucosamine residue adjacent to the non-reducing end of thegalactose residue participating in the β-galactosidic linkage and havingno sulfate group at its 6-position, the enzyme does not act on theβ-galactosidic linkage.

The enzyme group (2) preferably comprises endo-β-galactosidases.

The enzymes of the enzyme group (2) include, for example, anendo-β-galactosidase from Pseudomonas sp. IFO-13309 (K. Nakazawa, N.Suzuki, S. Suzuki, J. Biol. Chem., 250, 905 (1975); K. Nakazawa, S.Suzuki, J. Biol. Chem., 250, 912 (1975)) and an endo-β-galactosidaseproduced by Pseudomonas reptilivora disclosed in Japanese PatentPublication No. 57-41236 (1982) (herein these enzymes are sometimesreferred to simply as “keratanase” or “KSase”), which are preferred.

Enzyme group (3): Enzymes having the following activity and substratespecificity:

(a) Activity:

The enzyme cleaves a β-N-acetylglucosaminidic linkage of keratansulfate.

(b) Substrate specificity:

If sulfate groups are present at 6-position of an N-acetylglucosamineresidue participating in the β-N-acetylglucosaminidic linkage and at6-position of a galactose residue adjacent to a non-reducing end of theN-acetylglucosamine residue, respectively, the enzyme acts on theβ-N-acetylglucosaminidic linkage.

If a sulfate group is present at the 6-position of theN-acetylglucosamine residue participating in theβ-N-acetylglucosaminidic linkage but it is absent at the 6-position ofthe galactose residue adjacent to the non-reducing end of theN-acetylglucosamine residue, the enzyme acts on theβ-N-acetylglucosaminidic linkage.

If a sulfate group is present neither at the 6-position of theN-acetylglucosamine residue participating in theβ-N-acetylglucosaminidic linkage nor at the 6-position of the galactoseresidue adjacent to the non-reducing end of the N-acetylglucosamineresidue, the enzyme does not act on the β-N-acetylglucosaminidiclinkage.

The enzyme group (3) preferably comprises endo-βN-acetylglucosamidase.

The enzymes of the enzyme group (3) include, for example, anendo-β-N-acetylglucosamidase from Bacillus sp. Ks36 (Shinichi Hashimoto,Kiyoshi Morikawa, Hiroshi Kikuchi, Keiichi Yoshida, Kiyochika Tokuyasu,Seikagaku, 60, 935 (1988); herein sometimes referred to simply as“keratanase II” or “KSase II”) and an endo-β-N-acetylglucosamidase fromBacillus circulans KsT202 (disclosed in WO96/16166), which arepreferred.

Both of the enzyme groups (1) and (2) include endo-β-galactosidase typeenzymes but they have different substrate specificities depending onsulfate groups in the sugar chain. The enzyme group (3) includesendo-β-N-acetylglucosaminidase type enzymes. Therefore, the reducing endof an oligosaccharide obtained by enzymatic digestion is a galactoseresidue (Gal) when an endo-β-galactosidase type enzyme is used or anN-acetylglucosamine residue (GlcNAc) when anendo-β-N-acetylglucosaminidase type enzyme is used.

The substrate specificities of the enzyme groups (1) to (3) aresummarized below. It is indicated whether or not the enzyme cleaves aglycosidic linkage represented by “˜” in general formulae (a) to (k)below (i.e., β-galactosidic linkage in the case of the enzyme groups (1)and (2) or β-N-acetyl-glucosaminidic linkage in the case of the enzymegroup (3)).

TABLE 1 Enzyme Group (1) (a) . . . GlcNAc-Gal(6S)˜GlcNAc . . . Notcleave (b) . . . GlcNAc(6S)-Gal(6S)˜GlcNAc . . . Not cleave (c) . . .GlcNAc(6S)-Gal˜GlcNAc . . . Cleave (d) . . . GlcNAc-Gal˜GlcNAc . . .Cleave Enzyme Group (2) (e) . . . GlcNAc-Gal(6S)˜GlcNAc . . . Not cleave(f) . . . GlcNAc(6S)-Gal(6S)˜GlcNAc . . . Not cleave (g) . . .GlcNAc(6S)-Gal˜GlcNAc . . . Cleave (h) . . . GlcNAc-Gal˜GlcNAc . . . Notcleave Enzyme Group (3) (i) . . . Gal(6S)-GlcNAc(6S)˜Gal . . . Cleave(j) . . . Gal-GlcNAc(6S)˜Gal . . . Cleave (k) . . . Gal-GlcNAc˜Gal . . .Not cleave

In the above formulae, Gal represents a galactose residue, GlcNAcrepresents an N-acetylglucosamine residue, (6S) indicates that thehydroxyl group at the 6-position is sulfated, “−” and “˜” represent eacha glycosidic linkage, and “ . . . ” represents a structure of Gal andGlcNAc alternately linked through a glycosidic linkage.

(4) Conditions for Enzymatic Action

Keratan sulfate from cartilage has a high content of a disulfatedN-acetyllactosamine (LacNAc-diS) residue. In keratan sulfate from shark,its content is particularly high. Therefore, enzymes of the enzymegroups (1) and (2) (e.g., E-Galase or KSase) do not substantially act onsuch keratan sulfate. On the contrary, such keratan sulfate issubstantially degraded by the enzymes of the enzyme group (3) (e.g.,KSase II) and readily converted into disaccharides and tetrasaccharides.However, this keratan sulfate whose sugar chain has been partiallydesulfated is more susceptible to the action of the enzymes of theenzyme group (1) and (2) but in contrast less susceptible to the actionof the enzymes of the enzyme group (3) according as the degree ofdesulfation increases. Combination of the substrate specificity of theenzymes described above with the degree of partial desulfation ofkeratan sulfate permits control of the molecular size of anN-acetyllactosamine oligosaccharide to be finally obtained by the methodof the present invention. In other words, if the degree of partialsulfation is controlled such that the action of enzyme is increaseddepending on the specificity of the enzyme used, the molecular size willbe decreased. Conversely, if the degree of partial sulfation iscontrolled such that the action of the enzyme is decreased, themolecular size will be increased.

If the substrate is in small amounts, keratan sulfate with an adjustedsulfate group content and one or more enzymes of any one of the enzymegroups (1) to (3) above may be put together so that the enzyme orenzymes can act on the keratan sulfate. However, when the enzymetreatment is carried out on an large scale, it is preferred that theenzyme or enzymes is or are allowed to act continuously by usingimmobilized enzyme(s) obtained by binding the enzyme or enzymes to anappropriate solid phase (beads or the like) or a membrane type reactorwith an ultrafiltration membrane, a dialysis membrane or the like.

As stated above, a sulfated N-acetyllactosamine oligosaccharide having adesired molecular size can be obtained by appropriately combining thedegree of partial desulfation of keratan sulfate and the substratespecificity of a keratan sulfate-degrading enzyme.

(5) Fractionation of Sulfated N-acetyllactosamine Oligosaccharide

The fractionation of the sulfated N-acetyllactosamine oligosaccharideproduced by the action of the enzyme as described above may be performedusing conventional techniques for sugar chain separation andpurification. For example, the fractionation may be carried out byoperations such as adsorption chromatography, anion exchangechromatography, hydrophobic chromatography, gel filtration, gelpermeation chromatography, filter paper electrophoresis, filter paperchromatography, fractionation with organic solvents, or combinations ofthese. However, it is not limited thereto.

It is preferred that the sulfated N-acetyllactosamine oligosaccharidegenerated by the enzyme treatment be fractionated according to themolecular size of oligosaccharides. The fractionation method accordingto the molecular size is not limited particularly. For example, therecan be cited fractionation methods using gel filtration chromatographywith various gel filtration carriers or fractionation methods withorganic solvents. The gel filtration carrier is not limited particularlyas far as it is a carrier fabricated for molecular sieve and includescrosslinked acrylamide and crosslinked particles of polysaccharides suchas dextran, agarose, and cellulose and the like. Also, carriers with asuitable degree of crosslinking may be selected suitably depending onthe target molecular size of oligosaccharides. Use of gel filtrationchromatography may permit separation according to the molecular size ofthe respective oligosaccharides as far as oligosaccharides up tooctasaccharides are concerned, whereas separability of oligosaccharideshaving a chain length larger than the octasaccharides may decrease withan increased molecular size of the oligosaccharide. In this case,fractionation gives a mixture of oligosaccharides with several molecularsizes.

The mixture of oligosaccharides may be fractionated preliminarily withan anion exchange resin according to the density of sulfate group. Thisincreases the separability attained by subsequent gel filtrationchromatography. Polysulfated oligosaccharides, when they have the samesulfate group density, are adsorbed more strongly on anion exchangeresins as their molecular size becomes larger, so that they can beseparated from each other based on this principle by controlling theconcentration of salts.

In the case of fractionation with an organic solvent, it is preferred touse alcohol or acetone, for example.

The skeletons of sugar chain of the thus obtained sulfatedN-acetyllactosamine oligosaccharides are represented by general formulae(1) to (6) below. More specifically, general formulae (1) and (2) belowrepresent the skeletons of sulfated N-acetyllactosamine oligosaccharideobtained when one or more enzymes of the enzyme group (1) and/or (2) isor are allowed to act on the keratan sulfate with an adjusted sulfategroup content in the method of the present invention. On the other hand,general formulae (3) and (4) below represent the skeletons of sulfatedN-acetyllactosamine oligosaccharide obtained when one or more enzymes ofthe enzyme group (3) is or are allowed to act on the keratan sulfatewith an adjusted sulfate group content in the method of the presentinvention. Further, general formulae (5) and (6) below represent theskeletons of sulfated N-acetyllactosamine oligosaccharide obtained whenone or more enzymes of the enzyme group (1) and/or (2) and one or moreenzymes of the enzyme group (3) are allowed to act on the keratansulfate with an adjusted sulfate group content in the method of thepresent invention.

(GlcNAc-Gal)_(n)  (1)

SA-Gal-(GlcNAc-Gal)_(m)  (2)

(Gal-GlcNAc)_(n)  (3)

SA-(Gal-GlcNAc)_(m)  (4)

Gal-(GlcNAc-Gal)_(n)  (5)

GlcNAc-(Gal-GlcNAc)_(n)  (6)

In the formulae above, Gal represents a galactose residue, GlcNAcrepresents an N-acetylglucosamine residue, SA represents a sialic acidresidue, — represents a glycosidic linkage, n is an integer of 1 to 6,and m is an integer of 1 to 10.

For example, in the case where keratanase II, which is an enzymebelonging to the enzyme group (3) above, is allowed to act on keratansulfate with an adjusted sulfate group content in the method of thepresent invention, sulfated N-acetyllactosamine oligosaccharides havingthe skeletons represented by the following formulae are obtained:

Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (7)

Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (8)

 Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (9)

Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-GalGlcNAc-Gal-GlcNAc  (10)

SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (11)

SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (12)

SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (13)

SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (14)

wherein, Gal represents a galactose residue, GlcNAc represents anN-acetylglucosamine residue, SA represents a sialic acid residue, and -represents a glycosidic linkage.

The sulfated N-acetyllactosamine oligosaccharides having the skeletonsrepresented by the formulae (7) to (14) above are merely specificexamples of sulfated N-acetyllactosamine oligosaccharides having theskeletons represented by general formulae (3) and (4) above, and thepresent invention is not limited thereto.

Complete desulfation of the sulfated N-acetyllactosamineoligosaccharides having the skeletons represented by general formulae(1) to (6) above by the method described below gives rise toN-acetyllactosamine oligosaccharides represented by the followinggeneral formulae:

 (GlcNAc-Gal)_(n)  (1)

SA-Gal-(GlcNAc-Gal)_(m)  (2)

(Gal-GlcNAc)_(n)  (3)

SA-(Gal-GlcNAc)_(m)  (4)

Gal-(GlcNAc-Gal)_(n)  (5)

GlcNAc-(Gal-GlcNAc)_(n)  (6)

wherein, Gal represents a galactose residue, GlcNAc represents anN-acetylglucosamine residue, SA represents a sialic acid residue, —represents a glycosidic linkage, n is an integer of 1 to 6, and m is aninteger of 1 to 10.

For example, complete desulfation of the sulfated N-acetyllactosamineoligosaccharides having the skeletons represented by general formulae(7) to (14) above by the method described below gives rise toN-acetyllactosamine oligosaccharides represented by the followinggeneral formulae:

Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (15)

Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (16)

Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (17)

Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (18)

SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (19)

SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (20)

SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc  (21)

 SA-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlCNAc-Gal-GlcNAc-Gal-GlcNAc  (22)

wherein, Gal represents a galactose residue, GlcNAc represents anN-acetylglucosamine residue, SA represents a sialic acid residue, —represents a glycosidic linkage.

The N-acetyllactosamine oligosaccharides represented by the formulae(15) to (22) above are merely specific examples of N-acetyllactosamineoligosaccharides obtainable from sulfated N-acetyllactosamineoligosaccharides having the skeletons represented by general formulae(3) and (4) above, and the present invention is not limited thereto.

In the formulae (1) to (6) above, n is an integer of 1 to 6. However,long chain N-acetyllactosamine oligosaccharides, for example, those inwhich n is an integer of 3 to 6 are preferred, with those in which n isan integer of 5 or 6 being particularly preferred.

Also, in the formulae (1) to (6) above, m is an integer of 1 to 10.However, long chain N-acetyllactosamine oligosaccharides, for example,those in which m is an integer of 3 to 10 are preferred, with those inwhich m is an integer of 5 to 10 being particularly preferred.

In the formulae (1) to (22) above, preferred sialic acid residue (SA) isan N-acetylneuraminic acid residue (NeuAc). The glycosidic linkagebetween a sialic acid residue (SA) and a galactose residue (Gal)adjacent to the reducing end of the sialic acid residue (SA) ispreferably an α-2,3 glycosidic linkage or α-2,6 glycosidic linkage. Theglycosidic linkage between a galactose residue (Gal) and anN-acetylglucosamine residue (GlcNAc) adjacent to the reducing end of thegalactose residue (Gal) is preferably a β1,4 glycosidic linkage.Further, the glycosidic linkage between an N-acetylglucosamine residue(GlcNAc) and a galactose residue (Gal) adjacent to the reducing end ofthe N-acetylglucosamine residue (GlcNAc) is preferably a β-1,3glycosidic linkage. As will be described concerning complete desulfationof a sulfated N-acetyllactosamine oligosaccharide, no report has beenmade on the conditions of desulfating a sulfated N-acetyllactosamineoligosaccharide having a sialic acid residue, which conditions have beenfound for the first time by the present inventors. In view of this,those particularly preferred among N-acetyllactosamine oligosaccharidesand sulfated N-acetyllactosamine oligosaccharides in the method of thepresent invention include N-acetyllactosamine oligosaccharides andsulfated N-acetyllactosamine oligosaccharides that have a sialic acidresidue (for example, those represented by the formulae (2), (4), (11),(12), (13), (14), (19), (20), (21), (22) among (1) to (22)). (6)Complete desulfation of sulfated N-acetyllactosamine oligosaccharide

Complete desulfation of a sulfated N-acetyllactosamine oligosaccharideobtained by enzymatic digestion can be performed according to the methodfor partially desulfating keratan sulfate described above by setting updesulfation conditions appropriately so that substantially all thesulfate groups are desulfated. The desulfation conditions may varygreatly depending on the position at which sulfate groups are attached,the kind and type of linkage of sugars. In other words, sugar chainscould decompose or sulfate groups could remain depending on thetemperature and solvent conditions. In particular, when completedesulfation of a sulfated N-acetyllactosamine oligosaccharide having asialic acid residue is contemplated, too hard desulfation conditionswill result in removal of sialic acid residues in addition tosubstantially complete removal of sulfate groups. To prevent this, it isnecessary to set up conditions for complete desulfation while retainingsialic acid residues. There has been no report on such conditions.Therefore, as far as sulfated N-acetyllactosamine oligosaccharideshaving a sialic acid residue are concerned, the desulfation conditions(that is, conditions for complete desulfation while retaining sialicacid residues) described below have been elucidated for the first timeby the present inventors. Accordingly, the present invention includes amethod for completely desulfating a sulfated N-acetyllactosamineoligosaccharide having a sialic acid residue while retaining the sialicacid residue.

As the method for complete desulfation of a sulfated N-acetyllactosamineoligosaccharide, there can be cited, for example, acid hydrolysis,alkali decomposition, heating in an organic solvent, a method using asilylating agent, a method using an enzyme, as in the case of partialdesulfation of keratan sulfate. However, the reaction conditions aredifferent from those of partial desulfation.

Hereafter, the method for complete desulfation will be described morespecifically referring to a “dimethyl sulfoxide method” as an example ofthe desulfation method using an organic solvent, a“methanol-hydrochloric acid method” as an example of the desulfationmethod involving acid hydrolysis, and a “trimethylsilylating agentmethod” as an example of the desulfation method using a silylatingagent.

(a) Dimethyl Sulfoxide (DMSO) Method

A sulfated N-acetyllactosamine oligosaccharide is dissolved in water andthe solution is passed through a strong cation exchange resin (H type)to remove free sulfate groups. Then the solution is neutralized withpyridine to form pyridinium salt of the sulfated N-acetyllactosamineoligosaccharide. The pyridinium salt is dissolved in a DMSO solutioncontaining 5 to 20% by volume, preferably 10% by volume of water andheated at 70 to 100° C. for 2 to 10 hours, preferably at 100° C. for 5hours, to perform complete desulfation without causing decomposition ofthe sugar chain.

(b) Methanol-hydrochloric Acid Method

Hydrogen chloride gas is blown into dry methanol that is substantiallyfree of water to form methanol-hydrochloric acid solution. This solutionis diluted with dry methanol to adjust to 0.01 N to 1 N, preferably 0.1N. Well dried sulfated N-acetyllactosamine oligosaccharide is dispersedin the methanol-hydrochloric acid solution and stirred at 4 to 30° C.for 0.5 to 50 hours, preferably at room temperature for 15 hours, toeffect complete desulfation.

(c) Trimethylsilylating Agent (TMS) Method

This method is to desulfate a sulfated N-acetyllactosamineoligosaccharide by heating it together with anN-,O-bis(trimethylsilyl)acetamide (BTSA). For example,N-acetyllactosamine oligosaccharide is dissolved in a pyridine solutionand BTSA is added thereto in a ratio of 5 to 16 moles, preferably 10moles, per mole of sulfate group, followed by reaction at 40 to 90° C.for 0.5 to 3 hours, preferably 80° C. for 1 hour, to effect completedesulfation. The desulfation ratio obtained by this method, according tothe study by the present inventors, was confirmed to be freelycontrollable by varying temperature and treatment time.

Other methods for complete desulfation include, for example, thosemethods using an acid such as sulfuric acid, an alkali such as sodiumhydroxide.

Note that the method for complete desulfation is not limited to thosedescribed above.

Complete desulfation of a sulfated N-acetyllactosamine oligosaccharidegives rise to an N-acetyllactosamine oligosaccharide.

(7) Fractionation of N-acetyllactosamine Oligosaccharide

Fractionation of N-acetyllactosamine oligosaccharide can be carried outby use of conventional techniques of separation and purification ofsugar chains as in the above-described fractionation method for sulfatedN-acetyllactosamine oligosaccharide. For example, the fractionation maybe carried out by operations such as adsorption chromatography, anionexchange chromatography, hydrophobic chromatography, gel filtration, gelpermeation chromatography, filter paper electrophoresis, filter paperchromatography, fractionation with organic solvents, or combinations ofthese. However, it is not limited thereto.

The separation of an N-acetyllactosamine oligosaccharide having a sialicacid residue (sialyl N-acetyllactosamine oligosaccharide) from anN-acetyllactosamine oligosaccharide is not limited to a particularmethod and may be carried out by ion exchange chromatography. Nolimitation is posed on the method for separating N-acetyllactosamineoligosaccharides having different molecular sizes from each other. Forexample, separation by chromatography using a reverse phase base columnmay be carried out by increasing the concentration of methanol from 0 to20%.

An N-acetyllactosamine oligosaccharide, which is the oligosaccharide ofthe present invention, can be produced by the above-described methods.The oligosaccharide of the present invention will be described later on.

The method of the present invention preferably comprises the steps of:adjusting a sulfate group content of keratan sulfate, treating thekeratan sulfate having an adjusted sulfate group content with one ormore enzymes belonging to the enzyme groups (1) to (3) above to obtain asulfated N-acetyllactosamine oligosaccharide (step 1), and completelydesulfating the sulfated N-acetyllactosamine oligosaccharide (step 2).The step 1 preferably includes the step of fractionating the sulfatedN-acetyllactosamine oligosaccharide and the step 2 preferably includesthe step of fractionating the N-acetyllactosamine oligosaccharide. Thesefractionating steps may be included in only one of the steps (1) and(2). However, since fractionation is easier if a sulfate group ispresent, it is more preferred that the step 1 includes the step offractionating the sulfated N-acetyllactosamine oligosaccharide. It isparticularly preferred that the step 1 includes the step offractionating the sulfated N-acetyllactosamine oligosaccharide and thestep 2 includes the step of fractionating the N-acetyllactosamineoligosaccharide.

When the sulfated N-acetyllactosamine oligosaccharide is produced,partial desulfation or sulfation of keratan sulfate and degradation ofkeratan sulfate with an adjusted sulfate group content may be carriedout simultaneously (for example, in the same reactor) in the case wherethe enzyme having an activity of cleaving the glycosidic linkage ofkeratan sulfate will not be deactivated under the conditions for partialdesulfation or sulfation of keratan sulfate.

According to the method of the present invention, conventionallyobtainable keratan sulfate is partially desulfated in advance based onthe substrate specificity of keratan sulfate-degrading enzyme,oligosaccharides having desired molecular sizes can be obtained moreefficiently than subjecting keratan sulfate as it is to the action ofthe above-described enzyme.

By allowing an exo type glycosidase which excises a non-reducing endsugar from an N-acetyllactosamine oligosaccharide to act on theN-acetyllactosamine oligosaccharide represented by the general formulae(1) to (6) produced by the method of the present invention, there can beobtained an N-acetyllactosamine oligosaccharide having the sugar residuenumber reduced by one. Specific examples thereof will be describedbelow.

Allowing an exo-N-acetylglucosamindase to act on the N-acetyllactosamineoligosaccharides represented by the general formulae (1) and (6), therecan be obtained N-acetyllactosamine oligosaccharides represented by thegeneral formulae (23) and (24), respectively.

(GlcNAc-Gal)_(n)(1)→Gal-(GlcNAc-Gal)_(n-1)  (23)

GlcNAc-(Gal-GlcNAc)_(n)(6)→(Gal-GlcNAc)_(n)  (24)

In the above formulae, Gal represents a galactose residue, GlcNAcrepresents an N-acetylglucosamine residue, — represents a glycosidiclinkage, and n is an integer of 2 to 6.

Allowing an exo-β-galactosidase to act on the N-acetyllactosamineoligosaccharides represented by the general formulae (3) and (5), therecan be obtained N-acetyllactosamine oligosaccharides represented by thegeneral formulae (25) and (26), respectively.

(Gal-GlcNAc)_(n)(3)→GlcNAc-(Gal-GlcNAc)_(n-1)  (25)

Gal-(GlcNAc-Gal)_(n)(5)→(GlcNAc-Gal)_(n)  (26)

In the above formulae, Gal represents a galactose residue, GlcNAcrepresents an N-acetylglucosamine residue, — represents a glycosidiclinkage, and n is an integer of 2 to 6.

Further, optional use of a sialidase, for example,N-acetylneuraminidase, can give rise to an N-acetyllactosamineoligosaccharide free of sialic acid residues.

2. Oligosaccharide of the Present Invention

The oligosaccharide of the present invention is an oligosaccharideobtained by the method described below and having the following physicaland chemical properties:

(1) Method:

A method for producing an N-acetyllactosamine oligosaccharide, whichcomprises the steps of:

adjusting a sulfate group content of keratan sulfate,

allowing a keratan sulfate-degrading enzyme having an ability to cleavea glycosidic linkage of keratan sulfate to act on the keratan sulfatewith the adjusted sulfate group content to obtain a product,

and then completely desulfating the product.

(2) Physical and Chemical Properties:

The galactose residue content measured by an anthrone method is 35 to50% by weight.

The nitrogen content measured by elemental analysis is 3 to 4% byweight.

The molecular weight measured by mass spectrometry is 1,000 to 2,200.

The adjustment of sulfate group content is performed preferably bypartial desulfation.

The above-described method preferably includes at least the followingsteps 1 and 2:

Step 1:

The step of adjusting a sulfate group content of keratan sulfate, andallowing one or more enzymes belonging to any one of the followingenzyme groups (1) to (3) to act on the keratan sulfate with the adjustedsulfate group content to obtain a sulfated oligosaccharide:

Enzyme group (1): Enzymes having the following activity and substratespecificity:

(a) Activity:

The enzyme cleaves a β-galactosidic linkage of keratan sulfate.

(b) Substrate specificity:

If a galactose residue participating in the β-galactosidic linkage has asulfate group at its 6-position, the enzyme does not act on theβ-galactosidic linkage.

Whether or not a sulfate group is present at 6-position of anN-acetylglucosamine residue adjacent to a non-reducing end of agalactose residue participating in the β-galactosidic linkage and havingno sulfate group at its 6-position, the enzyme acts on theβ-galactosidic linkage.

Enzyme group (2): Enzymes having the following activity and substratespecificity:

(a) Activity:

The enzyme cleaves a β-galactosidic linkage of keratan sulfate.

(b) Substrate specificity:

If a galactose residue participating in the β-galactosidic linkage has asulfate group at its 6-position, the enzyme does not act on theβ-galactosidic linkage.

If a sulfate group is present at 6-position of an N-acetylglucosamineresidue adjacent to a non-reducing end of a galactose residueparticipating in the β-galactosidic linkage and having no sulfate groupat its 6-position, the enzyme acts on the β-galactosidic linkage.

If a sulfate group is absent at the 6-position of theN-acetylglucosamine residue adjacent to the non-reducing end of thegalactose residue participating in the β-galactosidic linkage and havingno sulfate group at its 6-position, the enzyme does not act on theβ-galactosidic linkage.

Enzyme group (3): Enzymes having the following activity and substratespecificity:

(a) Activity:

The enzyme cleaves a β-N-acetylglucosaminidic linkage of keratansulfate.

(b) Substrate specificity:

If sulfate groups are present at 6-position of an N-acetylglucosamineresidue participating in the β-N-acetylglucosaminidic linkage and at6-position of a galactose residue adjacent to a non-reducing end of theN-acetylglucosamine residue, respectively, the enzyme acts on theβ-N-acetylglucosaminidic linkage.

If a sulfate group is present at the 6-position of theN-acetylglucosamine residue participating in theβ-N-acetylglucosaminidic linkage but it is absent at the 6-position ofthe galactose residue adjacent to the non-reducing end of theN-acetylglucosamine residue, the enzyme acts on theβ-N-acetylglucosaminidic linkage.

If a sulfate group is present neither at the 6-position of theN-acetylglucosamine residue participating in theβ-N-acetylglucosaminidic linkage nor at the 6-position of the galactoseresidue adjacent to the non-reducing end of the N-acetylglucosamineresidue, the enzyme does not act on the β-N-acetylglucosaminidiclinkage.

Step 2:

The step of completely desulfating the sulfated oligosaccharide.

The enzyme belonging to the enzyme group (1) above is preferably anendo-β-galactosidase, for example, an endo-β-galactosidase fromEscherichia freundii (H. Nakagawa, T. Yamada, J-L. Chien, A. Gardas, M.Kitamikado, S-C. Li, Y-T. Li, J. Biol. Chem., 255, 5955 (1980); hereinsometimes referred to simply as “E-Galase”), which is preferred.

The enzyme belonging to the enzyme group (2) is preferably anendo-β-galactosidase, examples of which include an endo-β-galactosidasefrom Pseudomonas sp. IFO-13309 (K. Nakazawa, N. Suzuki, S. Suzuki, J.Biol. Chem., 250, 905 (1975); K. Nakazawa, S. Suzuki, J. Biol. Chem.,250, 912 (1975)) and an endo-β-galactosidase produced by Pseudomonasreptilivora disclosed in Japanese Patent Publication No. 57-41236 (1982)(herein these enzymes are sometimes referred to simply as “keratanase”or “KSase”), which are preferred.

The enzyme belonging to the enzyme group (3) is preferably anendo-β-N-acetylglucosaminidase, examples of which include anendo-β-N-acetylglucosaminidase from Bacillus sp. Ks36 (ShinichiHashimoto, Kiyoshi Morikawa, Hiroshi Kikuchi, Keiichi Yoshida, KiyochikaTokuyasu, Seikagaku, 60, 935 (1988); herein sometimes referred to simplyas “keratanase II” or “KSase II”) and an endo-β-N-acetylglucosaminidasefrom Bacillus circulans KsT202 (disclosed in WO96/16166), which arepreferred.

More preferably, the oligosaccharide of the present invention is onehaving a sialic acid content of 10 to 20% by weight as measured by athiobarbituric acid method in addition to the above-described physicaland chemical properties.

Further, the present invention provides a novel N-acetyllactosamineoligosaccharide represented by any one of the following general formulae(1) to (6):

(GlcNAc-Gal)_(n)  (1)

SA-Gal-(GlcNAc-Gal)_(m)  (2)

(Gal-GlcNAc)_(n)  (3)

SA-(Gal-GlcNAc)_(m)  (4)

Gal-(GlcNAc-Gal)_(n)  (5)

GlcNAc-(Gal-GlcNAc)_(n)  (6)

wherein, Gal represents a galactose residue, GlcNAc represents anN-acetylglucosamine residue, SA represents a sialic acid residue, —represents a glycosidic linkage, n is an integer of 1 to 6, and m is aninteger of 1 to 10, with a proviso that n is other than 1 in theformulae (1) and (3).

The method for producing the oligosaccharide of the present invention isnot limited to specific ones as far as the oligosaccharide of thepresent invention is obtained. However, it is preferred that it isproduced by the method of the present invention. The method of thepresent invention is described in the foregoing.

When the oligosaccharide of the present invention is produced by themethod of the present invention, for example, if it is contemplated toobtain the N-acetyllactosamine oligosaccharide represented by theformulae (1) or (2), an endo-β-galactosidase type enzyme (for example,an enzyme belonging to the enzyme group (1) or (2)) may be used in themethod of the present invention.

When it is contemplated to obtain the N-acetyllactosamineoligosaccharide represented by the formulae (3) or (4), anendo-β-N-acetylglucosaminidase type enzyme (for example, an enzymebelonging to the enzyme group (3)) may be used in the method of thepresent invention.

Further, when it is contemplated to obtain the N-acetyllactosamineoligosaccharide represented by the formulae (5) or (6), both of anendo-β-galactosidase type enzyme (for example, an enzyme belonging tothe enzyme group (1) or (2)) and an endo-β-N-acetylglucosaminidase typeenzyme (for example, an enzyme belonging to the enzyme group (3)) may beused in the method of the present invention.

In the case of the oligosaccharide of the present invention having asialic acid residue (for example, those represented by the formulae (2)and (4)), the sialic acid residue includes an N-acetylneuraminic acidresidue and an N-glycolylneuraminic acid residue. The sialic acidresidue is preferably an N-acetylneuraminic acid residue (NeuAc).

In the formulae (1) to (6) above, n is an integer of 1 to 6 (providedthat n is other than 1 in the formulae (1) and (3)). However, long chainN-acetyllactosamine oligosaccharides, for example, those in which n isan integer of 3 to 6 are preferred, with those in which n is an integerof 5 or 6 being particularly preferred.

In the formulae (1) to (6) above, m is an integer of 1 to 10. However,long chain N-acetyllactosamine oligosaccharides, for example, those inwhich m is an integer of 3 to 10 are preferred, with those in which m isan integer of 5 to 10 being particularly preferred.

The glycosidic linkage between a sialic acid residue (SA) and agalactose residue (Gal) adjacent to the reducing end of the sialic acidresidue (SA) is preferably an α-2,3 glycosidic linkage or α-2,6glycosidic linkage. The glycosidic linkage between a galactose residue(Gal) and an N-acetylglucosamine residue (GlcNAc) adjacent to thereducing end of the galactose residue (Gal) is preferably a β-1,4glycosidic linkage. Further, the glycosidic linkage between anN-acetylglucosamine residue (GlcNAc) and a galactose residue (Gal)adjacent to the reducing end of the N-acetylglucosamine residue (GlcNAc)is preferably a β-1,3 glycosidic linkage. As described concerningcomplete desulfation of a sulfated N-acetyllactosamine oligosaccharide,no report has been made on the conditions of desulfating a sulfatedN-acetyllactosamine oligosaccharide having a sialic acid residue, whichconditions have been found for the first time by the present inventors.In view of this, those particularly preferred among N-acetyllactosamineoligosaccharides in the method of the present invention andoligosaccharides of the present invention include N-acetyllactosamineoligosaccharides that have a sialic acid residue (for example, thoserepresented by the formulae (2) and (4) among (1) to (6)).

The sugar composition of the oligosaccharide of the present inventioncan be analyzed, for example, by an anthrone method (determination ofthe galactose residue), amino sugar analysis (determination of theN-acetylglucosamine residue), or a thiobarbituric acid method (TBAmethod; determination of the sialic acid residue). However, it is notlimited thereto. Also, the sulfate group content can be analyzed bydecomposing the oligosaccharide of the present invention withhydrochloric acid and analyzing by ion chromatography. However, it isnot limited to this method. The method for analyzing the molecular sizeof the oligosaccharide of the present invention is not limited toparticular ones. For example, it can be examined by analysis after gelfiltration chromatography and oligosaccharide after E-Galase treatment.The sugar chain sequence of the oligosaccharide of the present inventioncan be examined, for example, by a successive degradation method usingglycosidases.

The oligosaccharide of the present invention can be identified usingthese analytical methods, which methods can be used also for confirmingthe oligosaccharide of the present invention obtained, for example, bythe method of the present invention.

To the oligosaccharide of the present invention may optionally be added,for example, a sialic acid residue, a fucosyl residue, a sulfate group,ceramide or the like. Although these substances may be added by achemical method, it is preferred that they be added using an enzyme.

For example, addition of a fucosyl residue to the oligosaccharide of thepresent invention may be achieved by use of fucosyltransferase totransfer a fucosyl residue to the oligosaccharide of the presentinvention. On this occasion, it is preferred that the fucosyl residue bebonded to the N-acetylglucosamine residue, more preferably through anα-1,3 glycosidic linkage. Therefore, use of fucosyltransferase havingsuch an activity is preferred.

When it is contemplated to add a sialic acid residue to theoligosaccharide of the present invention represented by the formulae(1), (3), (5) or (6) above, sialyltransferase may be used to transfer asialic acid residue to the oligosaccharide of the present invention. Onthis occasion, it is preferred that the sialic acid residue be bonded tothe galactose residue, more preferably to the galactose residue on thenon-reducing end through an α-2,3 glycosidic linkage or an α-2,6glycosidic linkage. Accordingly, use of sialyltransferase having such anactivity is preferred.

Further, when it is contemplated to add a sulfate group to theoliqosaccharide of the present invention, sulfotransferase may be usedto transfer a sulfate group to the oligosaccharide of the presentinvention. As the sulfotransferase, there can be mentioned, for example,chondroitin 6-sulfotransferase (J. Biol. Chem., 268 (29), 21968-21974,(1993)).

Also, when addition of ceramide is contemplated, it is preferred to joinceramide to the reducing end of the oligosaccharides of the presentinvention.

These enzymes, which may be selected appropriately depending on thetarget oligosaccharide, can be used alone or a plurality of enzymes maybe allowed to act simultaneously. Alternatively, a plurality of enzymesmay be allowed to act successively.

EXAMPLES

Hereafter, the present invention will be described by examples.

Example 1

<1> Preparation of Sulfated N-Acetyllactosamine Oligosaccharide

1-1. Partial desulfation of keratan sulfate by a methanol-hydrochloricacid method and preparation of sulfated N-acetyllactosamineoligosaccharide

Keratan sulfate (hereafter, also referred to as “KS”) derived from sharkcartilage was one prepared according to a literature (Seikagaku, 33,746-752 (1961)). This contained 1.7 moles of sulfate group per mole ofthe constituent disaccharide.

Well dried KS (5 g) was suspended in 1 liter of anhydrous methanolsolution containing 5 ml of acetyl chloride (equivalent to 0.06 N HCl),and the suspension was allowed to react at 4° C. with shaking. After 0,2.5, 5, 10, 20, and 40 hours from the initiation of the reaction, 200 mleach of reaction mixtures were collected. Each sample was centrifuged tocollect insolubles, and the insolubles were then dissolved in distilledwater. The resulting solution was neutralized with 0.01 N sodiumhydroxide, dialyzed, desalted, and concentrated to 50 ml. Then, ethanolwas added thereto in an amount of 5 fold by volume to form precipitate,and then, the precipitate was collected. The precipitate was washedsuccessively with ethanol and with ether to collect dry powder. After 0,2.5, 5, 10, 20, and 40 hours from the initiation of the reaction, thereaction product had a sulfate group in an amount of 1.7, 1.3, 1.2, 1.0,0.9, and 0.8 mole, respectively, per mole of the constituentdisaccharide.

To each obtained dry powder (100 mg) was added 10 ml of 20 mM acetatebuffer (pH 6.5) to dissolve it. To the solution was added 1 unit ofkeratanase II (KSase II, manufactured by Seikagaku Corporation; 1 unit(U) is the amount of enzyme that liberates 1 μmole of a reducing group(N-acetylglucosamine) from keratan sulfate (derived from oxen cornea;manufactured by Seikagaku Corporation) at pH 6.5 and at 37° C. for 1hour.), and enzymatic digestion was carried out at 37° C. for 12 hours.To the solution after the enzymatic digestion, NaCl was added to a finalconcentration of 0.2 M, and the solution was applied to CellulofineGCL-90 sf (manufactured by Seikagaku Corporation) column (2×90 cm) andgel filtration was conducted with 0.2 M NaCl. The eluted sulfatedN-acetyllactosamine oligosaccharide was detected by an anthrone method(Extra issue, Tanpakushitsu, Kakusan, Koso, Seibutsu Kagaku Jikkenho XI,-Toshitsu Jikkenho-, p.15 (1968), Kyoritsu Shuppan). FIG. 1 illustratesthe relationship between the time of partial desulfation of KS by themethanol-hydrochloric acid method and the yield of sulfatedN-acetyllactosamine oligosaccharides of 6- to 12-saccharides. The yieldof sulfated N-acetyllactosamine oligosaccharides of 6- to 12-saccharideswas calculated as a ratio of the area occupied by the fractioncorresponding to the sulfated N-acetyllactosamine oligosaccharides of 6-to 12-saccharides to the total area of the figure surrounded by theelution curve of sulfated N-acetyllactosamine oligosaccharides obtainedby gel filtration and by the axis of time.

As illustrated in FIG. 1, when partial desulfation of KS was notconducted (that is, the reaction time by the methanol-hydrochloric acidmethod is 0 hour), the yield of the sulfated N-acetyllactosamineoligosaccharides of 6- to 12-saccharides was about 10%. In contrast,when the partial desulfation of KS by the methanol-hydrochloric acidmethod was conducted at 4° C. within 40 hours, the yield of the sulfatedN-acetyllactosamine oligosaccharides of 6- to 12-saccharides increasedabout 2 to 4 folds.

In particular, when the time of partial desulfating was 2.5 to 20 hours,the yield was at least about 30% and when the time of partialdesulfating was 5 to 10 hours, the yield reached at least about 40%.From this it will be readily understood by those skilled in the art thatwhen it is contemplated to obtain sulfated N-acetyllactosamineoligosaccharides of 6- to 12-saccharides by enzymatic digestion of KSusing 1 U of KSase II at 37° C. for 12 hours, the time of partialdesulfation of KS by the methanol-hydrochloric acid method at 4° C. ispreferably utmost 40 hours (except 0 hour), more preferably 2.5 to 20hours, and particularly preferably 5 to 10 hours.

1-2. Partial desulfation of keratan sulfate by a DMSO method andpreparation of sulfated N-acetyllactosamine oligosaccharide

A solution of KS (1 g) in water (40 ml) was passed through a column ofDowex 50WX (H⁺, 100-200 mesh; manufactured by Dow Chemical), and thenneutralized with pyridine to form the pyridinium salt, and waslyophilized. The yield of the lyophilized product was 980 mg.

The lyophilized product of KS pyridinium salt (800 mg) was dissolved in80 ml of 90% DMSO (10% H₂O). The resulting solution was divided intofour 20 ml-aliquots. Each was allowed to react at 40° C., 50° C., 60°C., 70° C. or 80° C. for 3 hours and then the same volume of water wasadded to stop the reaction. Each reaction solution was dialyzed in a tapwater overnight, concentrated to 4 ml, and then divided into 2 halves by2 ml. To one of them was added 0.5 ml of 250 mM acetate buffer (pH 6)containing 10 mM calcium acetate and to the other was added 0.5 ml of100 mM acetate buffer (pH 6.5). After adding an endo-β-galactosidasederived from Escherichia freundii (E-Galase, manufactured by SeikagakuCorporation) to the former and KSase II to the latter, each in an amountof 1 U (1 U is the amount of enzyme that liberates a reducing groupcorresponding to 1 μmole of galactose from keratan sulfate at pH 5.8 andat 37° C. for 1 minute), enzymatic digestion was carried out at 37° C.for 5 hours. The solutions after the enzymatic digestion were subjectedto gel filtration in the same manner as in 1-1 above and the resultingsulfated N-acetyllactosamine oligosaccharides were fractionated (FIG.3). FIG. 2 illustrates the relationship between the temperature ofpartial desulfation of KS by the DMSO method and the yield of sulfatedN-acetyllactosamine oligosaccharides of 6- to 12-saccharides. The yieldof sulfated N-acetyllactosamine oligosaccharides of 6- to 12-saccharideswas calculated in the same manner as in 1-1 above.

As illustrated in FIG. 2, in the case where an endo-β-galactosidasederived from Escherichia freundii is used in the enzymatic digestion,the yield of sulfated N-acetyllactosamine oligosaccharides of 6- to12-saccharides was at least about 20% when the temperature ofFpartialdesulfation of KS by the DMSO method was about 50 to 70° C., andabout 40% when the partial desulfation temperature was 60° C. In thecase where KSase II is used in the enzymatic digestion, the yield ofsulfated N-acetyllactosamine oligosaccharides of 6- to 12-saccharidesare at least about 20% when the temperature of partial desulfation of KSby the DMSO method was at least about 65° C., and reached to 30% whenthe partial desulfation temperature was 70° C., and about 40% when thepartial desulfation temperature was 80° C. From these results, it willbe readily understood by those skilled in the art that when it iscontemplated to obtain sulfated N-acetyllactosamine oligosaccharides of6- to 12-saccharides by the enzymatic digestion of KS using 1 U ofendo-β-galactosidase derived from Escherichia freundii at 37° C. for 5hours, the temperature at which partial desulfation of KS is performedby the DMSO method for 3 hours is preferably about 50 to 70° C., morepreferably 60° C. Also, it will be readily under stood by those skilledin the art that when it is contemplated to obtain sulfatedN-acetyllactosamine oligosaccharides of 6- to 12-saccharides by theenzymatic digestion of KS using 1 U of KSase II at 37° C. for 5 hours,the temperature at which partial desulfation of KS is performed by theDMSO method for 3 hours is preferably at least about 65° C., morepreferably at least 70° C., and particularly preferably at 80° C.

The reaction products (KS with an adjusted sulfate group content)obtained by partial desulfation of KS by the DMSO method for 3 hours at40, 50, 60, 70 and 80° C. had a sulfate group in a ratio of 1.6 moles,1.4 moles, 1.0 mole, 0.8 mole and 0.3 mole, respectively, per mole ofthe constituent disaccharide.

<2> Complete desulfation of sulfated N-acetyllactosamine oligosaccharideand fractionation of N-acetyllactosamine oligosaccharide 2-1. Completedesulfation of sulfated N-acetyllactosamine oligosaccharide

In the same manner as in 1-1 above, dried KS (10 g) was allowed to reactwith methanol-hydrochloric acid for 10 hours, followed by treatments byKSase II digestion and gel filtration fractionation, and the obtainedfractions corresponding to sulfated N-acetyllactosamine oligosaccharidesof 6- to 12-saccharides were separated, desalted, lyophilized, andcollected. The yield was 3.8 g.

The dry powder (3 g) was suspended in 100 ml of 0.01 N hydrochloricacid-methanol solution and slowly stirred at room temperature for 20hours. After the reaction was completed, the solution was neutralizedwith 0.01 N sodium hydroxide, the solvent was removed by evaporation,and then water was added thereto, followed by desalting using aCellulofine GCL-25 (manufactured by Seikagaku Corporation) column andlyophilizing to obtain N-acetyllactosamine oligosaccharides. The yieldwas 2.5 g (galactose: 32.4%, sialic acid: 1.8%).

2-2. Fractionation of N-acetyllactosamine oligosaccharide

The lyophilized powder (2 g) of N-acetyllactosamine oligosaccharidesobtained in 2-1 above was dissolved in 50 ml of water while heating andthe resulting solution was passed through a column of Dowex 1×8 Cl⁻,200-400 mesh (3×50 cm; manufactured by Dow Chemical) previouslyequilibrated with 5 mM NaCl solution and the column was washed with 1liter of the same solution. Then elution was performed by linearconcentration gradient formed by 5 mM to 200 mM NaCl (2 liters),followed by detection of galactose and sialic acid by an anthrone methodand a thiobarbituric acid method, respectively, to collect positivefractions. The anthrone-positive fractions in the solution that passedthrough the column without adsorption were concentrated and subjected togel filtration using Cellulofine GCL-90sf (manufactured by SeikagakuCorporation) column (4×100 cm) and water as a solvent. The eluate wasdetected by an anthrone method and peak portions corresponding to 6-,8-, 10-and 12-saccharides were collected, concentrated, desalted, andlyophilized (The yields were 253 mg for 6-saccharide, 324 mg for8-saccharide, 185 mg for 10-saccharide, and 54 mg for 12-saccharide). Ofthese, 100 mg of the 10-saccharide was dissolved in 10 ml of water andthe solution was applied to an ODS column, Daiso Pack SP-120-25-ODS-B(manufactured by Daiso Co., Ltd.; 2×50 cm) five times in an amount of 2ml each time. The column was eluted by use of a concentration gradientsystem from water to an aqueous 20% methanol solution. Then10-saccharide fraction was concentrated and lyophilized to collect it.The yield was 78 mg. Similarly, from 100 mg each of 8-saccharide and6-saccharide fractions were obtained 85 mg and 82 mg of purifiedpowders, respectively.

On the other hand, thiobarbituric acid-positive fractions that had beenadsorbed on an absorbent in a Dowex column, eluted with 2 liters ofaqueous 5 mM to 200 mM linear concentration gradient NaCl solution, andconcentrated were also applied to a Cellulofine GCL-90sf column in thesame manner as described above. Each of the fractions were subjected togel filtration with 0.2 M NaCl solution and fractions corresponding to9-saccharide, 11-saccharide, and 13-saccharide were collected, and eachof them was purified by repeating gel filtration using the same column,desalted, and lyophilized. As a result, sialyl N-acetyllactosamineoligosaccharides of 9-saccharide, 11-saccharide, and 13-saccharide(sialic acid residues are added to the non-reducing ends ofN-acetyllactosamine oligosaccharides) were obtained in amounts of 12 mg,15 mg, and 7 mg, respectively.

Of these, 6-saccharide, 8-saccharide, 10-saccharide, 9-saccharide,11-saccharide, and 13-saccharide fractions were measured for theirsialic acid contents, galactose contents, and nitrogen contents. Thesialic acid content, the galactose content, and the nitrogen contentwere measured by a thiobarbituric acid method, an anthrone method, andelemental analysis, respectively. Table 2 shows the results obtained.

TABLE 2 Sialic acid Galactose Nitrogen content content content (% byweight) (% by weight) (% by weight) 6-Saccharide — 46.2 3.538-Saccharide — 46.8 3.51 10-Saccharide — 46.9 3.54 9-Saccharide 16.838.5 3.75 11-Saccharide 13.4 40.1 3.72 13-Saccharide 11.8 41.3 3.70 (InTable 2, “—” indicates#no detection)

Further, mass spectrometric analysis was conducted on theabove-described 6-saccharide, 8-saccharide, 10-saccharide, and11-saccharide fractions. The mass spectrometric analysis was performedby fast atom bombardment mass spectrometry (FAB mass spectrometry).Table 3 shows main peak m/z values read from positive FAB mass spectraand negative FAB mass spectra. Table 3 also shows the molecular weightobtained from mass spectrometric analysis (“Found” in the table) and themolecular weight obtained based on the structural formula (“Calc.” inthe table).

TABLE 3 Positive Negative Molecular FAB Mass FAB Mass weight (m/z) (m/z)Found Calc. 6- 1114 [M + H]⁺ 1112 [M − H]⁻ 1113 1114 Saccharide 1136[M + Na]⁺ 8- 1479.1 [M + H]⁺ 1477.3 [M − H]⁻ 1478 1480 Saccharide 1501[M + Na]⁺ 10- 1844.7 [M + H]⁺ 1843.0 [M − H]⁻ 1843 1845 Saccharide1865.8 [M + Na]⁺ 11- 2136.7 [M + H]⁺ 2133.6 [M − H]⁻ 2136 2136Saccharide 2158.8 [M + Na]⁺ 2181.3 [M − H + 2Na]⁺

“Found” in Table 3 denotes molecular weights obtained by massspectrometric analyses.

“Calc.” in Table 3 denotes molecular weights obtained based on thefollowing structural formulae.

6-saccharide: (Gal-GlcNAc)₃

8-saccharide: (Gal-GlcNAc)₄

10-saccharide: (Gal-GlcNAc)₅

11-saccharide: NeuAc-(Gal-GlcNAc)₅

In the above formulae, Gal represents a galactose residue, GlcNAcrepresents an N-acetylglucosamine residue, NeuAc represents anN-acetylneuraminic acid residue, and—represents a glycosidic linkage.

The results shown in Table 3 confirmed that the molecular size(molecular weight) of the N-acetyllactosamine oligosaccharide obtainedaccording to the present invention substantially corresponded to thecalculated value. Further, it was confirmed that N-acetyllactosamineoligosaccharides with subtle differences in molecular size (molecularweight) could be fractionated depending on the molecular size.

From these results, it is suggested that among those shown in Table 2,the 9-saccharide would be NeuAc-(Gal-GlcNAc)₄ and the 13-saccharidewould be NeuAc-(Gal-GlcNAc₆.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides novelN-acetyllactosamine oligosaccharides and a novel method for producingN-acetyllactosamine oligosaccharides. The oligosaccharide of the presentinvention can be utilized as a raw material for synthesizing ligandsugar chains of selectin family that occurs only in trace amounts in thenature. The ligand sugar chains of selectin family are useful for anovel drug, particularly an anti-inflammatory agent.

According to the method 1 of the present invention, the oligosaccharideof the present invention can be produced on an industrial scale and atlow costs.

According to the method 2 of the present invention, sulfatedN-acetyllactosamine oligosaccharides, which are useful as intermediatesfor producing the oligosaccharide of the present invention, can beproduced on an industrial scale and at low costs. Further, it ispossible that the sulfated N-acetyllactosamine oligosaccharides have anovel physiological activity so that their application as a drug isexpected.

What is claimed is:
 1. An oligosaccharide obtained by a methodcomprising adjusting a sulfate group content of keratan sulfate,allowing an enzyme having an ability to cleave a glycosidic linkage ofkeratan sulfate to act on the keratan sulfate with the adjusted sulfategroup content to obtain a product, and then completely desulfating theproduct,wherein the oligosaccharide has the following physical andchemical properties: a galactose residue content measured by an anthronemethod is 35 to 50% by weight; a nitrogen content measured by elementalanalysis is 3 to 4% by weight; and a molecular weight measured by massspectrometry is 1,000 to 2,200, with the proviso that theoligosaccharide is not (Gal-GlcNAc)₃ or (Gal-GlcNAc)₄.
 2. Theoligosaccharide according to claim 1, wherein said method furthercomprises the following steps: (1) adjusting a sulfate group content ofkeratan sulfate, and allowing one or more enzymes belonging to any oneof the following enzyme groups (1) to (3) to act on the keratan sulfatewith the adjusted sulfate group content to obtain a sulfatedoligosaccharide, wherein the one or more enzymes have the followingactivities and substrate specificities: Enzyme group (1) consists ofenzymes which cleave a β-galactosidic linkage of keratan sulfate,wherein if a galactose residue participating in the β-galactosidiclinkage has a sulfate group at its 6-position, the enzyme does not acton the β-galactosidic linkage and, wherein whether or not a sulfategroup is present at 6-position of an N-acetylglucosamine residueadjacent to a non-reducing end of a galactose residue participating inthe β-galactosidic linkage and having no sulfate group at its6-position, the enzyme acts on the β-galactosidic linkage; Enzyme group(2) consists of enzymes which cleave a β-galactosidic linkage of keratansulfate, wherein if a galactose residue participating in theβ-galactosidic linkage has a sulfate group at its 6-position, the enzymedoes not act on the β-galactosidic linkage and, wherein if a sulfategroup is present at 6-position of an N-acetylglucosamine residueadjacent to a non-reducing end of a galactose residue participating inthe β-galactosidic linkage and having no sulfate group at its6-position, the enzyme acts on the β-galactosidic linkage and, whereinif a sulfate group is absent at the 6-position of theN-acetylglucosamine residue adjacent to the non-reducing end of thegalactose residue participating in the β-galactosidic linkage and havingno sulfate group at its 6-position, the enzyme does not act on theβ-galactosidic linkage; and Enzyme group (3) consists of enzymes whichcleave a β-N-acetylglucosaminidic linkage of keratan sulfate, wherein ifsulfate groups are present at 6-position of an N-acetylglucosamineresidue participating in the β-N-acetylglucosaminidic linkage and at6-position of a galactose residue adjacent to a non-reducing end of theN-acetylglucosamine residue, respectively, the enzyme acts on theβ-N-acetylglucosaminidic linkage and, wherein if a sulfate group ispresent at the 6-position of the N-acetylglucosamine residueparticipating in the β-N-acetylglucosaminidic linkage but it is absentat the 6-position of the galactose residue adjacent to the non-reducingend of the N-acetylglucosamine residue, the enzyme acts on theβ-N-acetylglucosaminidic linkage and, wherein if a sulfate group ispresent neither at the 6-position of the N-acetylglucosamine residueparticipating in the β-N-acetylglucosaminidic linkage nor at the6-position of the galactose residue adjacent to the non-reducing end ofthe N-acetylglucosamineresidue, the enzyme does not act on theβ-N-acetylglucosaminidic linkage; and (2) completely desulfating thesulfated oligosaccharide.
 3. An N-acetyllactosamine oligosacchariderepresented by any one of the following general formulae (1) to (6):(GlcNAc-Gal)_(n)  (1) SA-Gal-(GlcNAc-Gal)_(m)  (2) (Gal-GlcNAc)_(n)  (3)SA-(Gal-GlcNAc)_(m)  (4) Gal-(GlcNAc-Gal)_(n)  (5)GlcNAc-(Gal-GlcNAc)_(n)  (6) wherein Gal represents a galactose residue,GlcNAc represents an N-acetylglucosamine residue, SA represents a sialicacid residue, — represents a glycosidic linkage, n is an integer of 3 to6, and m is an integer of 3 to 10, with a proviso that n is 5 or 6 inthe formula (3).
 4. The oligosaccharide according to claim 1, whichcomprises a sialic acid residue.
 5. The oligosaccharide according toclaim 3, wherein n is 5 or
 6. 6. The oligosaccharide according to claim3, wherein m is an integer of 5 to 10.